Evaluation of acute and sub-chronic toxicity of Semelil (ANGIPARSTM), a new phytotherapeutic drug for wound healing in rodents

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Correspondence: [email protected]

Evaluation of acute and sub-chronic toxicity of Semelil

(ANGIPARS™), a new phytotherapeutic drug for wound healing in rodents

1

Abdollahi M,

²

Farzamfar B,

¹

Salari P,

^٭3

Khorram Khorshid HR,

⁴

Larijani B,

⁵

Farhadi M,

6

Madani SH

1Department of Toxicology & Pharmacology, Faculty of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, ²Pasteur Institute of Iran, Biotechnology Process Development Centre, Tehran, ³Genetic Research Centre, Social Welfare and Rehabilitation Sciences University, Tehran, ⁴Endocrinology and Metabolism Research Center (EMRC), Tehran University of Medical Sciences, Tehran, ⁵ENT - Head & Neck Surgery Department and Research

Center, Iran University of Medical Sciences, Tehran, ⁶Department of Biotechnology, Rabe Rashidi Institute, Tabriz, Iran

ABSTRACT

Semelil (ANGIPARS™), an herbal formulation containing Melilotus officinalis extract, is a novel compound being developed for treatment of chronic wounds, particularly diabetic foot ulcers. The purpose of this study was to investigate toxicological, pharmacological, and pathomorphological effects of I.M. and I.P. administration of Semelil in animals.

The acute toxicity parameters of Semelil diluted in normal saline (1:10 or 1:5) were determined after a single injection into BALB/c mice and Wistar rats in two steps. First, the LD50 was approximately assessed and then the precise lethal dose indices were estimated by the probit-analysis method. Specific single-dose effects of Semelil were monitored for clinical signs of toxicity, including general state of the animals, changes in their behavior, hematological and biochemical parameters for 14 days after drug administration. Then, subacute-chronic toxicity was evaluated in rats treated with Semelil for 3 months.

In acute toxicity study, the calculated LD50 for drug diluted at 1:5 was in the range of 44-52 ml/kg. The adverse effects at drug doses close to the LD50 included depressed mood, narcosis, and sleep. No adverse pharmacological or toxicological effects of the drug diluted at 1:10 and administered in the single-dose (25-50 ml/kg body wt.) or chronically (daily doses of 0.07 and 0.21 ml/kg body wt.) were noted. Thus, the animal studies demonstrated a favorable safety profile for the phytotherapeutic Semelil.

_________________________________________________________________________

Keywords: Semelil, ANGIPARS™, Preclinical trials, Rodents, Toxicity, LD50

INTRODUCTION

Among patients with diabetes mellitus, up to 15%

will experience a foot ulcer in their lifetime (1), and that is a major risk factor for future lower- extremity amputation in diabetics (2-4). In spite of all therapeutic efforts to prevent amputation in diabetic patients in the last decade, the incidence of lower-extremity amputation is rising both in developed and developing countries (5,6).

Therefore, new appropriate drugs to heal diabetic wound are essential. There are several recognized methods for treatment of sub-acute diabetic wounds. However, there is a need for alternative and complementary pharmacotherapy that provides accelerated healing and decrease the rate of recurrent ulcer complications and prevent major surgery and limb amputation. Herbal extracts are among medications with wound

healing properties that can be applied more easily on wounds. Previous studies have reported a potential usefulness of internal and external administration of Melilotus officinalis (Yellow Sweet Clover) extract for micro-circulation improvement in leg chronic venous insufficiency and hydrocortisone-like anti-inflammatory effects (7,8). The activity of extract of Herba Meliloti in combination with Dipteryx odorata and Ginkgo biloba was very significant for treatment of lymphedema of lower limbs (7-9). According to these known effects of Melilotus officinalis, a novel herbal-based compound was supposed to cure chronic wounds, particularly diabetic foot ulcers and was formulated. Advantageously, this drug “Semelil” have provided full healing with accelerated wound closure. Surprisingly, it also improved the quality of tissue in the healing

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wound with very noticeable hair growth on the scar tissue that ensured a lower rate of ulcer recurrence in the future.

The aim of this study was to evaluate safety of Semelil as a new candidate drug for wound healing in toxicity tests in rodent.

MATERIALS AND METHODS

Semelil herbal extract (ANGIPARS™) was prepared and delivered by ParsRoos Co. (Tehran, Iran). Total protein, creatinine, urea, total bilirubin, of lactate dehydrogenase and alkaline phosphatase activity were determined by a set of reagents of Diakom-Sinteco (Russia). The triglyceride and cholesterol levels were measured by a set of reagents of DiaSys Diagnostic Systems GmbH. (Germany). The activity of alanine aminoteransferase was determined using a set of reagents of Corway (Poland). All other biochemical parameters and enzyme activities were determined using a biochemical semiautomatic analyzer FP-901 (Labsystems, Finland). The blood elements were counted using automatic cell counter Picoskel (Hungary). The glucose level was measured by a set of reagents of the Labsystems Co. (Helsinki, Finland)

Drug solution preparation

ANGIPARS™ was freshly diluted in sterile saline (0.9% sodium chloride) at 1:5 or 1:10 ratios and then different portions of the solution were administered to animals.

Animals

BALB/c mice and Wistar rats were employed for administration of the preparation intramuscularly (in mice) and intraperitoneally (in mice and rats).

The protocol of the study was approved by the Center Animals Ethics Committee.

The animals, male and female BALB/c mice (18- 20g) and Wistar rats (120-180g) that had a veterinary and health certificates were taken from Laboratory animal farm Stolbovaya (RAMS, Russia) and the animals were kept in type T-3 (mice) and T-4 (rats) cages (Velaz, Prague, Czech Republic), 7-8 animals in each, at 20-22ºC and 60-65% humidity under a 12h light/dark cycle and had free access to food and tap water.

Single-dose toxicity evaluation

The toxicity of diluted Semelil was determined after a single injection into animals (male and female of 128 mice and 48 rats) in two steps.

First, the LD50 of Semelil intramuscularly (in mice) and intraperitoneally (in mice and rats) were approximately established with acute dose schedule used as recommended by Deichmann and LeBlanc (10). The precise fatal toxicity

indices of LD16, LD50 and LD84 were determined by the probit-analysis method of Litchfield and Wilcoxon (12). Then, single sub-acute doses of the drug were injected by I.M. and I.P. routs and the animals were observed for viability and clinical signs of toxicity on the day of dosing and then daily for 14 days (11).

Sub-chronic toxicity evaluation

Preliminary chronic safety studies with Semelil were performed on 90 male and female Wistar rats at sub-acute doses for 3 months. The rats were divided into 3 groups, 30 animals in each (15 male and 15 female) and Semelil was administered at doses of 0.07 ml/kg and 0.21 ml/kg once a day intramuscularly to the 2^nd and 3^rd groups, respectively. The first group received sterile solution of sodium chloride 0.9% as control. During the experiment, general state including the dynamics of body weight changes, appetite, motor activity, hair condition and behavior of the animals were recorded (13,14).

Blood samples (2.0-2.5 ml) were taken from caudal vein of each animal for hematological and biochemical analyses at the start of the study and 1 and 3 months after the first administration of drug or saline.

At the end of the chronic experiment, the animals’

euthanasia was performed by diethyl ether narcosis and pathomorphology of the internal organs and tissues of the animals were examined.

The autopsy of animals was performed immediately after death according to the pathoanatomic scheme, which reduced the possibility of autolysis.

Statistical Analyses

All measured variables were demonstrated as mean and SD. Statistical analyses were performed by using SPSS software, version 11.5 and Stata version 8. The probability levels of significance were based on Student’s t-test or non-parametric test, considering the normality of tested variable.

Statistically significance level was set at p<0.05.

RESULTS AND DISCUSSION Single dose toxicity evaluation

The results of acute intoxication study after single-dose of the drug introduced to BALB/c mice and Wistar rats and calculated I.M./I.P. fatal toxicity indices are presented in Table 1. The single i.m. injection of Semelildiluted in sterile saline at 1: 10 ratio at the doses of 0.5-1 ml/mouse (25-50 ml/kg) did not cause any toxicity and death. Increasing the dose to 1.5-2 ml/mouse (75- 100 ml/kg) remarkably decreased motor activity in mice and caused some deaths. The

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Table 1. Acute toxicity parameters of Semelil and ethanol 96% diluted (1:5) in solution of sodium chloride 0.9%

following I.M. or I.P. administration to mice and rats.

Fatal toxicity indices, ml/kg

Females Males

LD84

LD50±SD LD16

LD84

LD50±SD LD16

Routes of adminstration

BALB/c mice, Semelil

78.3 48.5±4.6

29.2 81.6

51.8±8.6 35.5

Intramuscular

74.7 44.9±5.3

25.6 77.5

47.7±4.9 28.4

Intraperitoneal

BALB/c mice, Ethanol 96%

42.7 35.2

28.3 44.8

37.3±2.1 30.4

Intraperitoneal

Wistar rats, Semelil

71.8 42.1±5.7

22.7 74.4

44.6±6.3 25.3

Intraperitoneal

The data are presented as mean ± standard deviation (SD); LD= lethal dose.

Table 2. Hematological parameters in male rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day. The control group received 0.9% sodium chloride.

Semelil 0.21 ml/kg Semelil

0.07 ml/kg Control

Observation period

Erythrocytes, 10¹²/l

7.4 ± 1.2 7.7 ± 1.2

7.5 ± 1.2 Before introduction

7.5 ± 1.6 7.3 ± 1.6

7.4 ± 1.6 1 month after introduction

7.4 ± 1.2 7.5 ± 1.2

7.6 ± 1.2 3 months after introduction

Leukocytes, 10⁹/l

11.9 ± 1.6 12.1 ± 1.6

11.7 ± 2 Before introduction

12.1 ± 1.2 12.6 ± 1.2

12.2 ± 2 12.3 ± 1.6

12.1 ± 2 3 months after introduction

Thrombocytes, 10⁹/l

685 ± 124 705 ± 88

692 ± 116 Before introduction

699 ± 104 693 ± 96

701 ± 124 1 month after introduction

707± 128 702 ± 124

697 ± 92 3 months after introduction

Hemoglobin, g/l

114 ± 12 111 ± 16

115 ± 20 116 ± 16

116 ± 16 114 ± 12

115± 16 3 months after introduction

Data are means ± standard deviation; All significance levels between groups were p>0.05.

intramuscular injection of Semelil diluted at 1:5 ratios was painful and caused profound depression and some deaths. In acute toxicity study, the calculated LD50 for drug diluted at 1:5 was in the range of 44-52 ml/kg. There was no significant difference (p > 0.05) between acute toxicity data obtained in experiments using I.M. and I.P. routes of drug administration (Table 1).

I.M. and I.P. injection of Semelil to BALB/c mice, in doses close to LD50, was accompanied by clear depression, narcosis and sleep in animals. In

the autopsy of animals plethora of the internal organs was observed. The clinical picture of the intoxication of Wistar rats with equivalent doses of Semelil at 1:5 dilution was similar to that of mice.

No specific and sex-related differences in the sensitivity of the animal species to the toxic effects of Semelil were detected when it was administered to BALB/c mice and Wistar rats intraperitoneally.

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Table 3. Hematological parameters in female rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day. The control group received 0.9% sodium chloride.

0.07 ml/kg Control

Observation period

Erythrocytes, 10¹²/l

7.4 ± 1.2 7.5 ± 1.6

7.3 ± 1.6 7.7 ± 1.2

7.5 ± 1.2 7.3 ± 1.2

Leukocytes, 10⁹/l

11.9 ± 1.6 12.5 ± 2.4

12.3 ± 1.2 12.3 ± 1.2

11.6 ± 1.2 11.9 ± 3.6

Thrombocytes, 10⁹/l

701 ± 92 692 ± 116

705 ± 104 699 ± 104

697 ± 116 705 ± 124

Hemoglobin, g/l

116 ± 16 114 ± 12

118 ± 12 116 ± 12

117 ± 12 118 ± 16

Table 4. Hepatic enzyme activity of male rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day. The control group received 0.9% sodium chloride.

Semelil 0.21 ml/kg Semelil 0.07 ml/kg

Control Observation period

Alkaline phosphatase, unit/l

516.8 ± 128 499.4 ± 154

514.9 ± 131 486.2 ± 175

480.8 ± 108 1 month after

introduction

494.4 ± 136 487.7 ± 145

469.7 ± 159 3 months after

introduction

Alanine aminotransferase, unit/l

61.84 ± 16 60.81 ± 22

66.29 ± 17 64.91 ± 18

introduction

62.71 ± 13 62.69 ± 20

introduction

Aspartate aminotransferase, unit/l

89.32 ± 26 91.38 ± 36

85.73 ± 36 84.44 ± 28

introduction

84.31 ± 35 86.66 ± 26

introduction

Lactate dehydrogenase, unit/l

798.36 ± 209 809.84 ± 165

771.44 ± 154 772.73 ± 190

820.26 ± 275 1 month after

introduction

797.36 ± 138 794.77 ± 246

introduction

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Body weight, female rats (3-month study)

0 50 100 150 200 250 300

0 2 4 6 8 10 12 14

Week

Weight (g)

Control ANGI (0.07ml/kg) ANGI (0.21ml/kg)

Table 5. Hepatic enzyme activity of female rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day . The control group received 0.9% sodium chloride.

0.07 ml/kg Control

Observation period

Alkaline phosphatase, unit/l

508.7 ± 192 501.4 ± 184

489.4 ± 156 558.1 ± 154

509.2 ± 154 1 month after introduction

491.9 ± 156 509.8 ± 153

Alanine aminotransferase, unit/l

62.08 ± 23 68.82 ± 19

68.78 ± 17 64.29 ± 23

59.89 ± 21 62.04 ± 21

Aspartate aminotransferase, unit/l

99.46 ± 30 100.88 ± 34

97.82 ± 33 102.76 ± 33

94.29 ± 36 108.27 ± 33

Lactate dehydrogenase, unit/l

902.35 ± 209 879.84 ± 205

863.76 ± 239 887.64 ± 186

875.24 ± 207 861.82 ± 153

Figure 1. The dynamics of body weight changes in female rats (3-month study) following Semelil I.M.

administration at the dose of 0.07 and 0.21 ml/kg once a day. The control group received 0.9% sodium chloride.

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Body weight, male rats (3-month study)

0 50 100 150 200 250 300 350 400

0 2 4 6 8 10 12 14

Week

Weight (g)

Control ANGI (0.07ml/kg) ANGI (0.21ml/kg)

Table 6.Biochemical parameters in male rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day . The control group received 0.9% sodium chloride.

0.07 ml/kg Control

Observation period

Total protein, g/l

62.8 ± 8 60.2 ± 14

66.7 ± 6.9 66.0 ± 10

64.4 ± 10.8 62.5 ± 13.7

Bilirubin, µmol/l

7.69 ± 1.5 7.89 ± 2.5

7.85 ± 2 7.80 ± 2

7.69 ± 1.8 7.78 ± 1.5

Urea, mmol/l

10.28 ± 3.5 11.81 ± 3.5

10.36 ± 2.5 10.47 ± 2.2

11.27 ± 2.6 10.72 ± 2.1

Creatinine, µmol/l

62.13 ± 21 61.75 ± 21

58.82± 22 Before introduction

63.52 ± 16.6 66.12 ± 22.6

69.69 ± 20.7 60.09 ± 16.6

Glucose, µmol/l

7.34 ± 1.0 7.58 ± 1.4

7.52 ± 1.2 7.33 ± 1.1

7.44 ± 0.9 7.47 ± 1.2

Total cholesterol, mmol/l

1.28 ± 0.76 1.14 ± 0.36

1.36 ± 0.56 1.04 ± 0.32

1.27 ± 0.32 1.16 ± 0.4

Triglycerides, mmol/l

0.70 ± 0.28 0.71 ± 0.32

0.67 ± 0.32 0.75 ± 0.48

0.79 ± 0.44 0.69 ± 0.24

Figure 2. The dynamics of body weight changes in male rats (3-month study) following Semelil I.M.

administration at the dose of 0.07 and 0.21 ml/kg once a day. The control group received 0.9% sodium chloride.

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Table 7. Biochemical parameters in female rats (3-month study) after Semelil I.M. administration at the dose of 0.07 and 0.21 ml/kg once a day . The control group received 0.9% sodium chloride.

0.07 ml/kg Control

Observation period

Total protein, g/l

66.4 ± 9.4 62.6 ± 10.7

66.8 ± 9.5 63.09 ± 11.3

64.5 ± 8.9 62.6 ± 9.6

62.9± 9.7 3 months after introduction

Bilirubin, µmol/l

7.59 ± 1.8 7.88 ± 1.9

7.48 ± 1.7 7.62 ± 1.9

7.69 ± 1.9 7.94 ± 2.1

Urea, mmol/l

10.87 ± 3 11.06 ± 2.7

10.29 ± 2.3 10.28 ± 2.5

10.20 ± 2 10.36 ± 2.2

Creatinine, µmol/l

62.18 ± 17.9 64.85 ± 23.2

65.23 ± 17.3 48.78 ± 19.4

61.41 ± 20.1 66.82 ± 18.5

Glucose, µmol/l

7.59 ± 1.4 7.39 ± 1.3

7.48 ± 1.2 7.58 ± 1.5

7.69 ± 1.2 7.49 ± 1.0

Total cholesterol, mmol/l

1.28 ± 0.32 1.09 ± 0.36

1.28 ± 0.36 1.28 ± 0.32

1.26 ± 0.36 1.19 ± 0.32

Triglycerides, mmol/l

0.71 ± 0.32 0.69 ± 0.28

0.78 ± 0.36 0.72 ± 0.36

0.79 ± 0.36 0.76 ± 0.36

Sub-chronic toxicity evaluation

Daily I.M. injections of Semelil at the doses of 0.07 and 0.21 ml/kg body weight had no effect on general state and behavior of rats. We did not detect any significant changes in hematological and biochemical parameters including total serum proteins, bilirubin, glucose content, triglycerides, cholesterol, and blood urea and creatinine, in test animal groups compared to control group after 1 and 3 months in rats. The activities of hepatic enzymes of test and control groups did not show any difference and corresponded to their physiological ranges, which are typical for these animals (Tables 1-7).

Mean body weights of rats in experimental groups, whether they received the preparation in doses of 0.07 or 0.21 ml/kg during all chronic

toxicity experiment, were not significantly different (p > 0.05) from those of control animals (Fig. 1,2).

Pathomorphological examination of animals after chronic experiment

At necropsy, macroscopic evaluation of the animals together with microscopic data showed that both test and control groups were practically healthy. No toxic or toxico-allergic effects of Semelil were revealed in the test groups (2^nd and 3^rd) of rats. No pathological changes of the internal organs and no local-irritating effects of the preparation were discovered in both test groups during the study period Our experiments showed that Semelil is a low-toxic substance after I.M. and I.P. administration to BALB/c mice and

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Wistar rats. Since there were no drug adverse effects and the calculated LD50 was in the range of 44-52 ml/kg in the acute dose study, it can be concluded that Semelil was well tolerated in rats both at I.M. administered doses of 0.07 and 0.21 ml/kg, which were used in the sub-acute chronic toxicity study.

Considering the profound effects of intraperitoneal and intramuscular injections of Semelil at dose levels close to the LD50 such as depression, narcosis and sleep, and their similarity to intoxication by ethanol (Table 1), which was used for the preparation of the drug, it can be assumed that these effects were directly related to this solvent.

The data indicated the stability of protein- productive function of liver, hepatic enzyme activity, carbohydrate metabolism, pancreas function, lipid metabolism, excretory kidney function of animals during the study period because we did not observe significant changes in

recorded hematological and biochemical parameters in test groups compared to control group.

CONCLUSION

Results of the present study gave an evidence of good tolerance of Semelil and the absence of undesirable effects on the functional state of the vital organs of the experimental animals in sub- acute and chronic test. Since antioxidants are believed to be useful in the management of diabetes and its complications (15) and regarding antioxidant potential of Semelil the next step of trials can be focused on diabetes.

ACKNOWLEDGEMENTS

The authors express their gratitude to ParsRoos Co. for providing Semelil (ANGIPARS™) used in the study and to Dr. M. H. Shahhosseiny , Dr.

V. Bakaev, and Dr. P. Hanachi for critically reading the manuscript and helpful comments.

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